ABSTRACT

The cry1-type genes of Bacillus thuringiensis represent the largest cry gene family, which contains 50 distinct holotypes. It is becoming more and more difficult to identify cry1-type genes using current methods because of the increasing number of cry1-type genes. In the present study, an improved PCR-restriction fragment length polymorphism (PCR-RFLP) method which can distinguish 41 holotypes of cry1-type genes was developed. This improved method was used to identify cry1-type genes in 20 B. thuringiensis strains that are toxic to lepidoptera. The results showed that the improved method can efficiently identify single and clustered cry1-type genes and can be used to evaluate cry1-type genes in novel strain collections of B. thuringiensis. Among the detected cry1-type genes, we identified four novel genes, cry1Ai, cry1Bb, cry1Ja, and cry1La. The bioassay results from the expressed products of the four novel cry genes showed that Cry1Ai2, Cry1Bb2, and Cry1Ja2 were highly toxic against Plutella xylostella, whereas Cry1La2 exhibited no activity. Moreover, Cry1Ai2 had good lethal activity against Ostrinia furnacalis, Hyphantria cunea, Chilo suppressalis, and Bombyx mori larvae and considerable weight loss activity against Helicoverpa armigera.

INTRODUCTION

Bacillus thuringiensis, which is a Gram-positive bacterium, is known for its specific toxicity toward insect pests (1). This toxicity is largely attributed to the insecticidal crystal proteins encoded by the cry genes (2–4). The cry1-type genes of B. thuringiensis are highly toxic to lepidopteran pests, and some genes have been used to develop plants with resistance to insect pests (5–7). Because of their potential application and commercial value, much research has focused on the discovery of novel cry1 genes; to date, approximately 258 cry1 genes have been cloned and named, and 50 distinct holotypes have been classified (http://www.lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/). Previous research has revealed that cry1 genes are typically found in clusters; for example, B. thuringiensis strains HD12 and HD525 contain at least four different cry1-type genes (8).

PCR is a simple and convenient investigative method and has been widely used to identify the vast variety of cry genes by the use of different primers (9–12). PCR-restriction fragment length polymorphism (PCR-RFLP) is a modified PCR technique that is generally used for the identification of known and unknown cry1-type genes (except for cry1I-type genes), and of parts of the cry7-type and cry9-type genes, according to the fragment lengths of digested PCR-amplified products described by Kuo and Chak (8). Some primers have been designed for the identification of cry1-type genes (13) and cry8-type genes (14) based on the PCR-RFLP method. However, it is becoming difficult to identify novel cry1-type genes using the PCR-RFLP method (8) because of the increase in the numbers of cry1-type genes.

To resolve this problem, an improved PCR-RFLP method was designed to directly identify the fourth class of cry1-type genes by dividing the cry1-type genes into four subgroups with relatively specific primers. In the present study, 22 B. thuringiensis strains were tested using the improved PCR-RFLP method; 2 of them, HD1 and HD29, were control strains, and others were isolated in our laboratory and are toxic to lepidoptera. The results showed that the improved PCR-RFLP method was effective and accurate at identifying cry1-type genes from B. thuringiensis strains.

DNA extraction.The B. thuringiensis strains were grown for 12 h on LB agar plates. Approximately 100 mg of cells (wet weight) was collected from the agar plates and washed with double-distilled water (ddH2O). DNA was prepared from these cells using the methods described by Song et al. (13) and was used as the DNA template for PCR-RFLP.

Improved PCR-RFLP.After personal computer (PC) gene multialignment analysis, four sets of primers were designed to divide the cry1-type genes into four subgroups; the sequences of these four oligonucleotide primers as well as the alignment between primers and all published cry1-type genes are shown in Table S1 in the supplemental material. PCR was performed in 50 μl containing 10 ng template DNA, 0.4 mmol/liter deoxynucleotide triphosphate, 0.2 μM (each) primer, and 1.5 U Taq DNA polymerase (TaKaRa Corporation, China) in reaction buffer. The amplification was performed as follows: 30 cycles of denaturation at 94°C for 1 min, annealing at 50°C for 1 min, and extension at 72°C for 2 min, which were followed by an additional extension at 72°C for 10 min. The PCR product was used for restriction endonuclease reactions, which were performed using a 20-μl mixture containing 10 μl PCR product and 1 U of the restriction endonuclease HinfI (according to analysis of restriction sites on the predicted DNA amplify fragment, HinfI could divide almost all cry1-type genes) in the appropriate reaction buffer. The PCR product was purified when nonspecific amplicons were present. The incubation temperature was set according to the manufacturer's instructions for the restriction endonuclease. The resulting restriction fragments were separated on 2% agarose gels. Table 1 shows the predicted fragment sizes of the PCR products for the known cry1-type genes.

Predicted sizes of PCR products and the RFLP fragments of various cry-type genes

Gene cloning and sequencing assay.For cloning the full-length cry1-type genes, primers were designed according to the reported genes (Table 2). PCR was performed using Pfu DNA polymerase (Tiangen, China) and a PTC-100 Peltier Thermal Cycler (MJ Research) as follows: 30 cycles of denaturation at 94°C for 60 s, annealing at 54°C for 50 s, and extension at 72°C for 4 min, which were followed by an additional extension at 72°C for 10 min. After amplification, the full-length PCR products were cloned into pSIMPLE-18 EcoRV/BAP vector (TaKaRa Corporation, China) for sequencing using an automated DNA sequencer (ABI-3730XL), and Vector NTI Suite 9 (Invitrogen; Carlsbad, CA) was used for sequence analysis.

The four novel cry1-type genes cloned from the wild-type B. thuringiensis strain and the primers for amplification of the full-length genes

Expression of cry1-type genes.To assay the toxicity of cry1-type genes, the genes were cloned and expressed in the E. coli Rosetta strain (DE3; Novagen). The primers listed in Table 2 were used for the amplification of full-length cry1-type genes. PCR was performed as described above. The products were cloned into the Ecl136II site of the pEB vector (16) and transformed into E. coli JM110 cells. Recombined plasmids containing in-frame cry1-type genes were transformed to the E. coli Rosseta strain (DE3). Expression was induced by the addition of IPTG (isopropyl β-d-1-thiogalactopyranoside) at a final concentration of 0.5 mmol/liter, and the cultures were incubated at 16°C at 220 rpm for 10 h. The cell pellet from a 10-ml culture of induced Rosetta (DE3) cells containing the recombinant expression vector was suspended in 1 ml of lysis buffer (20 mmol/liter Tris-HCl; pH 8.0) and sonicated (noise-isolating tamber; Ningbo Scientz Biotechnology Co., Ltd.) for 5 min (75% power; 3-s pulse on, 5-s pulse off). The lysate was centrifuged at 12,000 × g at 4°C for 20 min. The pellet (insoluble protein) was resuspended in 1 ml of lysis buffer (20 mmol/liter Tris-HCl; pH 8.0) and examined by SDS polyacrylamide gel electrophoresis (SDS-PAGE). ImageJ software (National Institutes of Health) was used to determine the intensity of the band against a bovine serum albumin (BSA) standard.

Insect bioassay.The insecticidal activities of the Cry1 proteins, which were tested against Ostrinia furnacalis, Helicoverpa armigera, Hyphantria cunea, and Chilo suppressalis, were assayed by exposing neonatal larvae to an artificial diet (17). Bioassays were performed in 24-well trays. Each well of the assay tray contained approximately 400 mg of diet. Each concentration was tested with 24 insects that were individually placed in each well, and the amount or total weight of the surviving insects was recorded after 4 days for H. cunea or 7 days for the others. Analysis of toxicity against Plutella xylostella second-instar larvae was conducted on fresh cabbage using the leaf-dip bioassay (18), and the number of surviving larvae was recorded after 2 days. Analysis of toxicity against B. mori neonate larvae was conducted on mulberry leaves also using the leaf-dip bioassay (18), and the number of surviving larvae was also recorded after 2 days. All bioassays were repeated at least three times using at least seven concentrations. The 50% lethal concentration (LC50) and 50% weight loss concentration (WLC50) values were calculated by Probit analysis (19).

Nucleotide sequence accession numbers.The sequences of the full-length cry1Bb and cry1Ja genes from WBT-2, the cry1Ai gene from SC6H8, and the cry1La gene from BtS6 have been deposited in the GenBank database, and the accession numbers are listed in Table 2.

RESULTS

Evaluation of the improved PCR-RFLP method.Two strains, HD1 and HD29, containing several known cry1-type genes, were used to evaluate the improved method.

An RFLP pattern with 0.72-, 0.65-, 0.56-, 0.39-, 0.35-, 0.33-, and 0.21-kb fragments was obtained for strain HD1 using primer set one (CRY1P1 and CRY1PR) (Fig. 1, lane 1 in HD1), which suggested that the HD1 strain contained cry1Aa, cry1Ab, and cry1Ac genes. RFLP patterns obtained using primer set four (CRY1P4 and CRY1PR) (Fig. 1, lane 4 in HD1) conformed to the predicted fragments of cry1Ia. There were no products identified using primer set two or three. Thus, the HD1 strain was determined to contain four cry1-type genes, cry1Aa, cry1Ab, cry1Ac, and cry1Ia, which corresponded with known results (10, 20).

Strain HD29 produced a cry1Ab gene RFLP pattern (Fig. 1, lane 1 in HD29) using primer set one (CRY1P1 and CRY1PR). RFLP patterns obtained using primer sets two (CRY1P2 and CRY1PR) (Fig. 1, lane 2 in HD29) and four (CRY1P4 and CRY1PR) (Fig. 1, lane 4 in HD29) suggested that the HD29 strain contained cry1Bd and cry1Ie genes. Using primer set three (CRY1P3 and CRY1PR), HD29 produced a novel RFLP pattern (Fig. 1, lane 3 in HD29) that contained fragments of 1.14, 0.93, 0.59, 0.35, 0.26, and 0.11 kb, which corresponded to the predicted fragments of cry1Db and cry1Gb genes. Thus, the HD29 strain was determined to contain five cry1-type genes: cry1Ab, cry1Bd, cry1Db, cry1Gb, and cry1Ie.

Identification of cry1-type genes in 20 native B. thuringiensis strains using the improved method.Twenty native B. thuringiensis strains (listed in Table 3) isolated by our laboratory were identified using the improved PCR-RFLP method. Among these strains, 17 contained reported cry1-type genes, which are shown in Table 3 (RFLP patterns not shown), whereas the other 3 B. thuringiensis strains, WBT-2, BtS6, and SC6H8, were determined to have novel cry1-type genes (listed in Table 2 and Table 3).

The strain WBT-2 RFLP pattern (Fig. 1, lane 1 in WBT-2), which was obtained using primer set one (CRY1P1 and CRY1PR), was the same as that obtained with the HD29 strain, which suggested that the WBT-2 strain contained the cry1Ab gene. The RFLP pattern (Fig. 1, lane 2 in WBT-2) obtained using primer set two (CRY1P2 and CRY1PR) contained fragments of 1.11, 0.22, and 0.15 kb, which corresponded to the main specific fragments of cry1Bb. The RFLP pattern (Fig. 1, lane 3 in WBT-2) obtained using primer set three (CRY1P3 and CRY1PR) contained fragments of 0.86, 0.70, 0.52, 0.36, 0.35, 0.28, 0.19, 0.17, 0.15, and 0.10 kb, which corresponded to the predicted fragments of cry1Da and cry1Ja. The RFLP pattern (Fig. 1, lane 4 in WBT-2) obtained using primer set four (CRY1P4 and CRY1PR) corresponded to the predicted fragments of the cry1Id gene. Thus, strain WBT-2 contains five cry1-type genes: cry1Ab, cry1Bb, cry1Da, cry1Id, and cry1Ja (Table 3).

The strain BtS6 RFLP pattern (Fig. 1, lane 1 in BtS6) obtained using primer set one (CRY1P1 and CRY1PR) contained fragments of 0.76, 0.72, 0.65, 0.57, 0.33, 0.31, 0.24, and 0.15 kb, which corresponded to the fragments predicted for the cry1La, cry1Aa, and cry1Ai genes. The RFLP pattern (Fig. 1, lane 2 in BtS6) observed using primer set two (CRY1P2 and CRY1PR) corresponded to the cry1Be gene. The RFLP pattern (Fig. 1, lane 3 in BtS6) obtained using primer set three (CRY1P3 and CRY1PR) contained fragments of 0.76, 0.72, 0.57, 0.33, 0.31, 0.24, and 0.15 kb, which corresponded to the predicted fragments of the cry1Ah and cry1Ea genes. The RFLP pattern (Fig. 1, lane 4 in BtS6) obtained using primer set four (CRY1P4 and CRY1PR) corresponded to the predicted fragments of the cry1Ia gene. Thus, BtS6 contains seven cry1-type genes: cry1Aa, cry1Ah, cry1Ai, cry1Be, cry1Ea, cry1Ia, and cry1La (Table 3).

The strain SC6H8 RFLP patterns (Fig. 1, lane 1 and 2 in SC6H8) obtained using primer sets one (CRY1P1 and CRY1PR) and two (CRY1P2 and CRY1PR) were similar to that obtained with BtS6 strain, which suggested that SC6H8 contains the cry1La, cry1Aa, cry1Ai, and cry1Be genes. The RFLP pattern (Fig. 1, lane 3 in SC6H8) obtained using primer set three (CRY1P3 and CRY1PR) corresponded to the predicted fragments of the cry1Ah gene. The RFLP pattern (Fig. 1, lane 4 in SC6H8) obtained using primer set four (CRY1P4 and CRY1PR) corresponded to the predicted fragments of the cry1Ia gene. Thus, SC6H8 contained six cry1-type genes: cry1Aa, cry1Ah, cry1Ai, cry1Be, cry1Ia, and cry1La (Table 3).

Cloning and sequencing assay of the cry1-type genes.The full-length cry1Bb and cry1Ja genes from WBT-2, cry1Ai gene from SC6H8, and cry1La gene from BtS6 were cloned into pSIMPLE-18 EcoRV/BAP vector and sequenced. cry1Ai, cry1Bb, cry1Ja, and cry1La are novel genes. Their sequences were submitted to the B. thuringiensis Delta-Endotoxin Nomenclature Committee, and logical names were assigned to their products as follows: Cry1Ai2, Cry1Bb2, Cry1Ja2, and Cry1La2, respectively.

Expression of cry1 genes.The cry1Ai, cry1Bb, cry1Ja, and cry1La genes were successfully expressed in the E. coli Rosetta (DE3) strain. SDS-PAGE analysis showed that all four genes could be expressed and produced products that were approximately 130 kDa (Fig. 2).

Toxicity bioassay.The results of the toxicity bioassay against P. xylostella are shown in Table 4. As seen with the Cry1Ac toxin, Cry1Ai2, Cry1Bb2, and Cry1Ja2 exhibited strong toxicity against P. xylostella larvae, with LC50s of 0.7 μg/ml, 0.32 μg/ml, and 1.01 μg/ml, respectively. However, neither weight loss nor mortality was detected with Cry1La2 even at concentrations up to 300 μg/ml.

DISCUSSION

The potential commercial value of new biopesticides developed from B. thuringiensis strains or transgenic insect-resistant plants containing cry genes has made the isolation and identification of these strains routine in many laboratories (1, 21). The use of specific- or multiple-primer PCR can identify cry genes efficiently (22, 23); however, this method is not suitable for the identification of new cry genes because of the high specificity of the amplifications. The PCR-RFLP method can identify known and novel genes based on the distinct patterns produced by digestion of the PCR products amplified using conserved primers. However, the increasing numbers of cry1-type genes make it challenging to utilize PCR-RFLP identification. To solve this problem, an improved PCR-RFLP method was introduced by grouping the cry1-type genes into four smaller subfamilies with relatively specific primers. According to the computer-predicted RFLP patterns, the improved PCR-RFLP method is able to effectively and accurately identify 41 cry1-holotype genes. The characterization of two strains with known cry1-type genes indicated that the improved PCR-RFLP method is an effective technique to reveal cry1-type genes. The characterization of 20 native B. thuringiensis strains suggested that the improved PCR-RFLP method is a valuable tool to evaluate the resource of cry1-type genes in B. thuringiensis strains. Moreover, it is suitable for single cry1-type gene identification and complex cry1-type gene separation.

Using the improved method, we found that the results from strain HD1 were in agreement with previous reports, whereas those from strain HD29 were not. Previous reports on the combination of cry-type genes in B. thuringiensis subsp. galleriae were controversial because this subspecies has been reported to contain cry1Da, cry9Aa, and cry7A genes (24), cry1Ab, cry1Ac, cry1Cb, and cry1Da genes (25), or cry1Aa, cry1Ca, cry1Cb, and cry1Fa genes (8). The reason for these diverse results might be that we focused on the identification of cry1-type genes using different primers; what is more, it may because of evolution of strains caused by plasmid transfer and gene recombination. Anyway, the PCR-RFLP method is used for the identification of toxin genes.

To evaluate the four novel cry1-type genes (cry1Ai2, cry1Bb2, cry1Ja2, and cry1La2), bioassays against P. xylostella were performed. Three Cry1 proteins, Cry1Ai2, Cry1Bb2, and Cry1Ja2, were highly toxic against P. xylostella (Table 4). Because the Cry1A toxin is widely used for the control of lepidopteran insects, we evaluated Cry1Ai2 toxicity against five other lepidopteran insects, and the results show that it has highly lethal toxicity to O. furnacalis, B. mori, H. cunea, and C. suppressalis and weight loss activity against H. armigera. The result indicated that Cry1Ai2 was a worthy candidate for control of lepidopteran insects. Also, the Cry1Ai2 toxicity pattern showed its worth in insecticidal mechanism research. The Cry1Ai2 protein shares 91% amino acid identity with the Cry1Ac toxin; however, the 9% difference makes the two toxins different with respect to toxicity against H. armigera and B. mori. Cry1Ai has highly lethal toxicity against B. mori and low activity against H. armigera, while Cry1Ac has highly lethal toxicity against H. armigera (26) and low activity against B. mori (27). The further research of Cry1Ai and Cry1Ac may help in understanding the molecular basis of specificity and in the design of new biopesticides.

In conclusion, an improved PCR-RFLP method was established. This method can be used to identify single and clustered cry1-holotype genes and to evaluate the number of cry1-type genes in B. thuringiensis strains regardless of whether the cry genes are known or novel.

ACKNOWLEDGMENTS

This study was supported by the National Natural Science Foundation of China (31272115), Key Project of Chinese National Programs for Fundamental Research and Development (973 Program, 2009CB118902), the National High Technology Research and Development Program of China (863 Program, 2011AA10A203), and the National Science and Technology Major Project (2013ZX08009003-001-004 and 2013ZX08004-004-009).

We thank Yingping Liang, Gemei Liang, Kanglai He (State Key Laboratory for Biology of Plant Diseases and Insect Pests), Yongping Huang (The Key Laboratory of Insect Developmental and Evolutionary Biology), and Yongan Zhang (The Key Laboratory of Forest Protection) for the insects supplied.